Abstract / Project Summary The overall objective of the proposed work is to understand how the Dmrt1 gene controls spermatogonial stem cell formation in the mammalian testis. The testis has two essential functions: production of sperm, the cells that serve as vehicles for the immortality of male germ line DNA; and production of hormones that direct other parts of the body to develop in a male-specific manner. Failures of these processes cause infertility, germ cell cancer, and disorders of sex development (DSD). Dmrt1 belongs to a family of conserved transcriptional regulators and it controls multiple crucial processes in the mammalian testis, both in germ cells and somatic cells. A recent discovery is that DMRT1 is required to form spermatogonial stem cells (SSCs). The central hypothesis of this proposal is that DMRT1 acts as a pioneer transcription factor to open chromatin and allow other cooperating transcription factors to bind and regulate gene expression, thereby orchestrating SSC formation. This proposal has three aims focused on a deeper understanding of how DMRT1 controls SSC cell fate. Aim 1 asks how DMRT1 directs SSC formation. It examines how loss of Dmrt1 affects key events in SSC formation including proliferation and expression of SSC regulators. It then seeks a mechanistic understanding of how DMRT1 regulates target gene transcription to control cell fate, testing the hypothesis that DMRT1 is a pioneer transcription factor. The experiments will employ an array of state-of-the- art genomic tools including ChIP-seq and ATAC-seq to find key regulatory targets and learn how DMRT1 binding in SSCs affects enhancer activity. Motif searches and ChIP will be used to identify likely cooperating transcription factors. Aim 2 will identify the genes regulated directly and indirectly by DMRT1 during SSC formation. Regulated transcripts will be identified by standard and single-cell RNA-seq and HiChIP will be used to link regulatory regions to one another and to the transcriptional start sites they control. Aim 3 will test the functional importance of selected DMRT1 cooperating transcription factors and target genes, using lentiviral knockdown in cultured SSCs followed by in vivo validation for top candidates. The proposed work has direct human health relevance: DMRT1 in humans is linked to infertility, testicular germ cell cancer and DSD including male-to-female sex reversal. As a result, the proposed work will help uncover the mechanistic basis of SSC formation and may provide general insights into stem cell biology.